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Removal of contaminating erythrocytes from human polymorphonuclear leucocyte preparations
Abstract:
A simple discontinuous gradient of Percoll has been used to remove the erythrocyte (RBC) contamination in human polymorphonuclear leucocyte (PMN) preparations avoiding the possible adverse effects of differential lysis. Mononuclear cells are also removed leaving a preparation with a white cell purity of more than 99% PMN contaminated by less than 2% RBC. The yield of PMN was 40% with respect to whole blood. The metabolic response of cells prepared in this way was found to be comparable with PMN isolated using the technique of Böyum [3]. Small differences in the iodination response of the purified PMN were shown to be the result of the removal of RBCs rather than an inherent effect of the Percoll isolation procedure.
Insights
This study introduces a Percoll gradient method for purifying polymorphonuclear leukocytes (PMN), achieving over 99% purity and avoiding adverse effects. The isolated PMN show comparable metabolic responses to those prepared using other methods.
Area of Science:
- Hematology
- Cell Biology
- Immunology
Background:
- Human polymorphonuclear leukocyte (PMN) isolation often faces challenges with erythrocyte (RBC) contamination.
- Conventional methods like differential lysis can cause adverse effects on PMN viability and function.
- Efficient and gentle isolation techniques are crucial for accurate downstream cellular analysis.
Purpose of the Study:
- To develop a simple Percoll gradient method for isolating pure human PMN.
- To evaluate the efficacy of this method in removing RBC and mononuclear cells.
- To compare the metabolic response of Percoll-isolated PMN with those obtained by established methods.
Main Methods:
- A discontinuous Percoll gradient was employed for PMN isolation.
- The method aimed to remove contaminating erythrocytes and mononuclear cells.
- Cell purity, yield, and metabolic responses were assessed.
Main Results:
- The Percoll gradient method yielded PMN preparations with >99% purity and <2% RBC contamination.
- PMN yield was 40% relative to whole blood.
- Metabolic responses of isolated PMN were comparable to those obtained using the Böyum technique.
- Observed differences in iodination response were attributed to RBC removal, not the Percoll procedure itself.
Conclusions:
- A simple discontinuous Percoll gradient effectively isolates highly pure human PMN.
- This method avoids adverse effects associated with differential lysis.
- Percoll-isolated PMN exhibit functional characteristics comparable to conventionally isolated cells, making it a suitable alternative for research.